Many differentiated but renewable cell types, including blood, skin, and sperm, are derived from dedicated precursor cells, or stem cells, which maintain the essentially unlimited capacity for continued division. Despite their biological medical importance, the mechanisms that control stem cell behavior are poorly understood. We proposed to identify molecular mechanisms that specify stem cell identity, self-renewal, and commitment to differentiation, using the Drosophila male germ line as a model tissue, then test if related mechanisms involving homologous genes regulated stem cell behavior in mammals. Variations of the mechanisms we uncover may govern stem cell behavior in other organisms and tissues, leading to applications in cancer, therapeutics, and genetic engineering. We have identified three genes required for normal stem cell behavior and fate in the Drosophila male germ line: zonder kloten (zk) acts in establishment and survival of male germ cells, one shot (osho) is required for germ line stem cell self-renewal, and stem cell tumor (stet) is required for commitment to or initiation of differentiation by germ line stem cell daughters. To elucidate the mechanism of action of these genes in specifying tissue renewal from dedicated stem cells, we will clone osho and stet, examine the predicted protein products for structural motifs and homology to proteins of known function, and determine the stage and cell types in which they are expressed. We will confirm if zk encodes an unusual nucleoporin expressed in gonads and the larval hematopoietic organs of Drosophila, determine if lethal alleles of zk effect hematopoiesis, and test the relationship of zk to importin and JAK kinase genes required for both hematopoiesis and male germ cell development in Drosophila. We will isolate the test if mouse homologues of zk and eventually osho and stet are expressed in and play roles in hematopoietic and/or male germ line stem cell behavior in mammals. Finally, we will use powerful first generation genetic selections for suppressor mutations in Drosophila to identify additional genes in the pathway controlling stem cell self-renewal.